This invention relates to treatment of antibiotic associated diarrhea, including Clostridium difficile associated diarrhea (CDAD) and pseudomembranous colitis (PMC) and other conditions associated with C. difficile infection. More specifically, the invention concerns neutralization of C. difficile toxin B, a cytotoxin associated with CDAD, PMC and other conditions caused by C. difficile. 
The following references are cited in the application as numbers in brackets ([]) at the relevant portion of the application.
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The disclosure of the above publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if the language of each individual publication, patent and patent application were specifically and individually included herein.
The anaerobic organism Clostridium difficile is the major causative agent of antibiotic-associated bacterial diarrhea and pseudomembranous colitis (PMC) among mainly elderly patients in hospitals and long term care facilities [1,2]. The organism cannot compete successfully with the normal microbial flora in the adult colon, but when the normal intestinal microflora is altered, for example by antibiotic treatment, C. difficile is able to colonize the gut in high numbers. Antibiotic therapy accounts for 98% of all cases of C. difficile associated diarrhea (CDAD). However, any predisposing condition which alters the normal intestinal flora, including any condition which requires extensive immunosuppressive treatment, can also lead to the development of CDAD. For example, recent evidence suggests that AIDS patients are also high risk candidates for acquiring CDAD [3,4].
C. difficile produces two exotoxins, toxin A (an enterotoxin) and toxin B (a cytotoxin) which appear to play important roles in causing CDAD. It has long been thought that toxin A is primarily responsible for the disease. It acts by binding to epithelial cells in the intestine, resulting in the destruction of these cells and causing the secretion of fluid into the intestine. The destruction of these protective epithelial cells by toxin A represents the crucial step leading to the development of diarrhea. Once damage has occurred to the epithelial cells, the potent cytotoxin B can then gain access to underlying sensitive tissues and initiate additional clinical symptoms [5-10,13,19-20,53-56,57-59,61-64]. However, in a recent in vitro study [46], toxin B was found to be more potent at damaging human colonic epithelium than toxin A, suggesting that toxin B may play a more important role in CDAD than previously believed.
Toxin A has been found to display a lectin-like activity which allows it to bind to an oligosaccharide receptor on epithelial cells. Several oligosaccharide sequences have been identified as potential receptors for toxin A [60,66]. The cellular receptor for toxin B has not been determined, but there are some indications that toxin B binds to erythrocytes [47, 48]. Steroids have also been proposed as potential receptors for toxin B [47].
The current therapy for patients who suffer from CDAD or PMC is to remove the offending drug and begin oral administration of the antibiotics Metronidazole or Vancomycin along with fluid replacement [3,14]. Vancomycin is only used in certain situations when patients cannot tolerate or are not responsive to Metronidazole treatment. Vancomycin is not used routinely because of its high cost and the possibility that its overuse may encourage the development of Vancomycin-resistant microorganisms. Metronidazole therapy is effective in about 80% of the patients who suffer from CDAD or PMC. In about 20% of patients, the diarrhea returns after discontinuing antibiotic treatment [15]. In such individuals, episodes continue to recur until the normal intestinal flora is reestablished and the number of C. difficile organisms is reduced. This is a slow process, since antibiotics such as Metronidazole, which disturb the balance of the normal intestinal flora, are administered each time the diarrhea occurs.
The only other treatment for CDAD and PMC which removes toxin activity from the intestinal tract involves the use of multigram quantities of anion exchange resins such as cholestyramine and colestipol given orally in combination with antibiotics. This approach has been used to treat mild to moderately ill patients, as well as individuals who suffer from multiple episodes of diarrhea [16,17]. This form of therapy has only been moderately successful in treating the disease [18]. In addition to their lack of efficacy, there are several other disadvantages associated with the use of ion exchange resins. Ion exchange resins do not bind specifically to toxin A or toxin B. Thus, ion exchange resins may also bind antibiotics, resulting in suboptimal levels of antibiotic within the gut. This can also occur with other medications patients may be receiving for unrelated conditions. A further disadvantage of ion exchange resins is the disagreeable lingering taste which is associated with oral administration of these compounds.
With respect to methods of diagnosis, one method for detecting C. difficile in a sample is to culture the sample. The disadvantages of this method include the length of time required to obtain a result and interference by non-pathogenic, i.e. non-toxin producing, C. difficile strains. Other methods involve the use of specific antisera or monoclonal antibodies. These methods are based on the detection of toxin A or toxin B in clinical samples. U.S. Pat. Nos. 4,863,852 and 5,098,826 describe methods for detecting C. difficile toxin A by the use of reagents containing biological receptors for toxin A, including the xcex1Gal(1-3)xcex2Gal(1-4)xcex2GlcNAc, X and Y antigen oligosaccharide sequences, bound to a support. U.S. Pat. No. 5,635,606 teaches that certain synthetic oligosaccharide sequences covalently attached to a biocompatible solid support, e.g., Chromosorb P(trademark), may be used to bind toxin A.
In view of the above, there is a need for an effective treatment for antibiotic associated diarrhea. In particular, a compound is needed which can neutralize C. difficile toxin B and/or both C. difficile toxin A and toxin B. A preferred compound would be administered noninvasively, such as orally, in a suitable pharmaceutical formulation.
The invention provides compositions and methods for the prevention and treatment of antibiotic associated diarrhea, pseudomembranous colitis and other conditions caused by Clostridium difficile toxin B.
In one aspect, the invention provides a method to bind and remove C. difficile toxin B from a sample suspected of containing said toxin B comprising contacting the sample with at least one toxin B binding oligosaccharide sequence covalently attached to an inert support through a non-peptidyl compatible linker arm under conditions wherein the toxin B is absorbed to the support; and separating the support containing the absorbed toxin B from the sample.
In another aspect, the invention provides a method to prevent or ameliorate one or more conditions mediated by C. difficile toxin B in a patient suffering from or susceptible to said condition, comprising administering to the patient an effective amount of a composition comprising at least one toxin B binding oligosaccharide sequence covalently attached to a pharmaceutically acceptable inert support through a non-peptidyl compatible linker arm, wherein said oligosaccharide sequence binds toxin B, and wherein the composition is capable of being eliminated from the gastrointestinal tract.
In a further aspect, the invention provides a pharmaceutical composition useful in treating or preventing CDAD and related conditions initiated by C. difficile toxin B, comprising at least one oligosaccharide sequence covalently attached to a pharmaceutically acceptable inert support through a non-peptidyl compatible linker arm, wherein said oligosaccharide sequence binds toxin B, and a pharmaceutically acceptable carrier, wherein said composition is capable of being eliminated from the gastrointestinal tract.
In yet another aspect, the invention provides a method to bind and remove C. difficile toxins A and B from a sample suspected of containing said toxins A and B comprising contacting the sample with at least one toxin A binding oligosaccharide sequence and at least one toxin B binding oligosaccharide sequence covalently attached to an inert support through a non-peptidyl compatible linker arm under conditions wherein the toxins are absorbed to the support; and separating the support containing the absorbed toxins from the sample.
In a still further aspect, the invention provides a method to prevent or ameliorate one or more conditions mediated by C. difficile toxins A and B in a patient suffering from or susceptible to said condition, comprising administering to the patient an effective amount of a composition comprising at least one toxin A binding oligosaccharide sequence and at least one toxin B binding oligosaccharide sequence covalently attached to a pharmaceutically acceptable inert support through a non-peptidyl compatible linker arm, wherein said oligosaccharide sequences bind toxins A and B, and wherein the composition is capable of being eliminated from the gastrointestinal tract.
In a yet further aspect, the invention provides a pharmaceutical composition useful in treating or preventing CDAD and related conditions initiated by C. difficile toxins A and B, comprising at least one oligosaccharide sequence covalently attached to a pharmaceutically acceptable inert support through a non-peptidyl compatible linker arm, wherein said oligosaccharide sequence(s) binds both toxin A and toxin B, and a pharmaceutically acceptable carrier, wherein said composition is capable of being eliminated from the gastrointestinal tract.